Type II anti-CRISPR proteins as a new tool for synthetic biology

Abstract

The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas (CRISPR-associated proteins) system represents, in prokaryotes, an adaptive and inheritable immune response against invading DNA. The discovery of anti-CRISPR proteins (Acrs), which are inhibitors of CRISPR-Cas, mainly encoded by phages and prophages, showed a co-evolution history between prokaryotes and phages. In the past decade, the CRISPR-Cas systems together with the corresponding Acrs have been turned into a genetic-engineering tool. Among the six types of CRISPR-Cas characterized so far, type II CRISPR-Cas system is the most popular in biotechnology. Here, we discuss about the discovery, the reported inhibitory mechanisms, and the applications in both gene editing and gene transcriptional regulation of type II Acrs. Moreover, we provide insights into future potential research and feasible applications.

Keywords: CRISPR-Cas system; Cas9; gene editing; synthetic biology; transcriptional regulation; type II anti-CRISPRs.

Figure 1.

Type II-A anti-CRISPR inhibitory mechanisms. Up to now, the inhibitory mechanism of Acrs have been understood only partially. In II-A subtype, AcrIIA1 triggers SpyCas9 degradation; AcrIIA2, A4, and A13 are capable of preventing SpyCas9-sgRNA complex from binding the DNA; AcrIIA6 inhibits St1Cas9 specifically by inducing St1Cas9-sgRNA complex dimerization; AcrIIA15 hinders sgRNA loading onto SauCas9 and keeps SauCas9 in an inactive state

Figure 2.

Type II-C anti-CRISPR inhibitory mechanisms. AcrIIC1 is capable of inactiving the nuclease activity of Cas9 by preventing the HNH nuclease domain rotation; AcrIIC2 hinders sgRNA loading onto Cas9; AcrIIC3 is able to bind apo-Cas9, Cas9-sgRNA duplex, and Cas9-sgRNA-DNA complex. Moreover, it inhibits Nme1Cas9 by trapping its HNH domain and inducing the dimerization of two independent Nme1Cas9s. AcrIIC4 and AcrIIC5 prevent DNA binding to the Cas9-sgRNA complex

Figure 3.

Scheme of the light-controllable AcrIIA4 OFF-switch CASANOVA. In dark conditions, the Acr-LOV variant retains the native function of AcrIIA4 and inhibits Cas9 RNP activity. Conversely, upon photoexcitation, Acr-LOV misfolds such that AcrIIA4 can no longer interact with Cas9 RNP. Hence, Cas9 RNP activity is fully recovered and DNA cleavage takes place [101]

Figure 4.

Scheme of the miR-122-responsive AcrIIA4 OFF-switch. miR-122 is produced specifically and in large amount in hepatocytes (here, hepatocellular carcinoma cells Huh-7). By inserting an miR-122 binding site in the 3ʹ UTR of the acrIIA4 transgene, AcrIIA4 translation is suppressed in Huh-7 cells but not in cells of different kind. Hence, cell-specific gene editing is achieved by exploiting cell-specific miRNAs [106]

Figure 5.

AcrIIA4-based synthetic gene circuits [110]. a) I1-FFL (Incoherent type 1 feedforward Loop). The expression of GFP and AcrIIA4 is activated by VPR-dCas9-sgRNA. The fluorescence signal increases until AcrIIA4 reaches a concentration that completely inhibits VPR-dCas9-sgRNA. From this moment on, GFP decays. Hence, the I1-FFL behaves as a pulse generator. b) Shield1-dependent circuit. Left panel: a destabilization domain (DD) is fused to AcrIIA4. As a result, the chimeric protein DD-AcrIIA4 is unstable and quickly degraded in the absence of Shield1. By binding Shield1, however, DD-AcrIIA4 folds into a stable configuration and escapes fast degradation. Right panel: only in the presence of Shield1, DD-AcrIIA4 binds and inactivates VPR-dCas9-sgRNA. Since fluorescence expression (output) demands the absence of Shield1 (input), the circuit corresponds to a NOT gate